The present invention relates to methods of detecting G protein-coupled receptor (GPCR) activity in vitro and in vivo. The present invention provides methods for identifying compounds that activate the GPCR regulatory pathway and methods for identifying ligands of GPCRs.
G protein-coupled receptors (GPCRs) are cell surface proteins that translate hormone or ligand binding into intracellular signals. GPCRs are found in all animals, insects, and plants. GPCR signaling plays a pivotal role in regulating various physiological functions including phototransduction, olfaction, neurotransmission, vascular tone, cardiac output, digestion, pain, and fluid and electrolyte balance. Although they are involved in various physiological functions, GPCRs share a number of common structural features. They contain seven membrane domains bridged by alternating intracellular and extracellular loops and an intracellular carboxyl-terminal tail of variable length.
The magnitude of the physiological responses controlled by GPCRs is linked to the balance between GPCR signaling and signal termination. The signaling of GPCRs is controlled by a family of intracellular proteins called arresting. Arrestins bind activated GPCRs, including those that have been agonist-activated and especially those that have been phosphorylated by G protein-coupled receptor kinases (GRKs).
Receptors, including GPCRs, have historically been targets for drug discovery and therapeutic agents because they bind ligands, hormones, and drugs with high specificity. Approximately fifty percent of the therapeutic drugs in use today target or interact directly with GPCRs. See eg., Jurgen Drews, (2000) “Drug Discovery: A Historical Perspective,” Science 287:1960-1964.
Although only several hundred human GPCRs are known, it is estimated that several thousand GPCRs exist in the human genome. Of these known GPCRs, many are orphan receptors that have yet to be associated with a function or ligands.
One method of assaying GPCR activity, as disclosed in U.S. Pat. Nos. 5,891,646, and 6,110,693, both to Barak et al., uses a cell expressing a GPCR and a conjugate of an arrestin and a detectable molecule.
Accordingly, there is a need to provide accurate, easy to interpret methods of detecting G protein-coupled receptor activity.